The genomes of higher organisms are highly annotated by specific chromosomal proteins and histone modifications along active genes, regulatory elements, or silent regions. An ongoing challenge is to decipher the rules that establish and maintain chromatin organization. Classic epigenetic regulators such as the Polycomb group (PcG), the Trithorax group (TrxG), and Heterochromatin Protein 1 (HP1), have profound roles in developmental control of the genome in all higher organisms examined. However, one obstacle to understanding their function has been the trade-off between removing them from the DNA, to allow purification, and the resultant loss of weak or transient interactions with key partners. Our new approach allows us to affinity-purify fragmented chromatin with proteins and RNAs attached, using cross-linking to avoid disruption of weak interactions. Using our approach, the linked DNA, protein, histone peptides, and RNA fractions can be separately analyzed using comprehensive sequencing and mass spectrometry. In Aim 1, a dual tag allows affinity purification from low amounts of relatively crude extracts, giving us the ability to look at early events in the establishment of these key chromatin-associated complexes. In Aim 2, we probe their molecular mechanisms by measuring their impact on RNA polymerase distribution across the genome at high resolution. Success in our studies will be relevant to understanding the establishment of conserved genome organization and function, which is central to normal human development and physiology. PUBLIC HEALTH RELEVANCE: Cellular genomes are organized into active and silent chromatin, characterized by specific DNA, RNA, and protein composition. Perturbation of this organization can lead to aberrant development and diseases such as cancer in humans. The goal of our studies is to understand the rules for establishment and maintenance of chromatin organization, and to document its precise functions in gene regulation.